Molecular Mechanisms of Endothelialitis in SARS-CoV-2 Infection: Evidence for VE-Cadherin Cleavage by ACE2.

Long COVID-19 syndrome appears after Severe Acute Respiratory Syndrome-Corona Virus (SARS-CoV-2) infection with acute damage to microcapillaries, microthrombi, and endothelialitis. However, the mechanisms involved in these processes remain to be elucidated. All blood vessels are lined with a monolayer of endothelial cells called vascular endothelium, which provides a the major function is to prevent coagulation. A component of endothelial cell junctions is VE-cadherin, which is responsible for maintaining the integrity of the vessels through homophilic interactions of its Ca++-dependent adhesive extracellular domain. Here we provide the first evidence that VE-cadherin is a target in vitro for ACE2 cleavage because its extracellular domain (hrVE-ED) contains two amino acid sequences for ACE2 substrate recognition at the positions 256P-F257 and 321PMKP-325L. Indeed, incubation of hrVE-ED with the active ectopeptidase hrACE2 for 16 hrs in the presence of 10 µM ZnCl2 showed a dose-dependent (from 0.2 ng/µL to 2 ng/µL) decrease of the VE-cadherin immunoreactive band. In vivo, in the blood from patients having severe COVID-19 we detected a circulating form of ACE2 with an apparent molecular mass of 70 kDa, which was barely detectable in patients with mild COVID-19. Of importance, in the patients with severe COVID-19 disease, the presence of three soluble fragments of VE-cadherin (70, 62, 54 kDa) were detected using the antiEC1 antibody while only the 54 kDa fragment was present in patients with mild disease. Altogether, these data clearly support a role for ACE2 to cleave VE-cadherin, which leads to potential biomarkers of SARS-CoV-2 infection related with the vascular disease in "Long COVID-19".

Dialogue between VE-Cadherin and Sphingosine 1 Phosphate Receptor1 (S1PR1) for Protecting Endothelial Functions.

The endothelial cells (EC) of established blood vessels in adults remain extraordinarily quiescent in the sense that they are not actively proliferating, but they fulfill the necessary role to control the permeability of their monolayer that lines the interior of blood vessels. The cell-cell junctions between ECs in the endothelium comprise tight junctions and adherens homotypic junctions, which are ubiquitous along the vascular tree. Adherens junctions are adhesive intercellular contacts that are crucial for the organization of the EC monolayer and its maintenance and regulation of normal microvascular function. The molecular components and underlying signaling pathways that control the association of adherens junctions have been described in the last few years. In contrast, the role that dysfunction of these adherens junctions has in contributing to human vascular disease remains an important open issue. Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid mediator found at high concentrations in blood which has important roles in the control of the vascular permeability, cell recruitment, and clotting that follow inflammatory processes. This role of S1P is achieved through a signaling pathway mediated through a family of G protein-coupled receptors designated as S1PR1. This review highlights novel evidence for a direct linkage between S1PR1 signaling and the mediation of EC cohesive properties that are controlled by VE-cadherin.

Microtumor Spheroids Provide a Model for Studying Molecules Involved in Vascular Organization: An Illustrative Study for VE-cadherin.

BACKGROUND/AIM: A growing body of research is contributing to the development of three-dimensional (3D) tissue models to close the gap between two-dimensional (2D) cell culture and animal models. Here, we report fundamental studies to confirm the modification of vascular endothelial (VE)-cadherin by a tumor microenvironment using 2D and 3D in vitro models of triple-negative breast cancer cells co-cultured with endothelial cells. MATERIALS AND METHODS: Breast cancer cells were cultivated as a monolayer (2D) on plates for 5 days or as microtumor spheroids (3D) with endothelial cells for up to 6 days. Phosphotyrosine-containing protein panels were analyzed in both cell types and upon co-culture. Microtumor spheroid size was evaluated via phase contrast microscopy. The content of VE-cadherin and phospho-VE-cadherin was determined. The effect of microtumor spheroid on the capillary network formed by endothelial cells was quantified by ImageJ Angiogenesis Analyzer. Sunitinib was used to determine drug efficacy in this model. RESULTS: The activity of signaling pathways in endothelial cells, including phosphorylation of Y685-VE-cadherin, was increased by the presence of breast cancer cells. In the 3D co-culture system, we established a ratio of the two cell types which allowed viability for 6 days. As a proof-of-concept of the 3D co-culture system for the process of drug discovery and development, we used the system to quantify the efficacy of sunitinib on the phosphorylation of VE-cadherin. CONCLUSION: In summary, we established 2D and 3D breast cancer-endothelial cell test systems compatible for detection of minimally tyrosine-phosphorylated proteins including VE-cadherin. The systems are capable of quantifying the effect of drugs on a tissue model of angiogenesis. This is a step towards developing tools for drug-efficacy testing that do not rely on live animals.

Persistence of RNA transcription during DNA replication delays duplication of transcription start sites until G2/M.

As transcription and replication use DNA as substrate, conflicts between transcription and replication can occur, leading to genome instability with direct consequences for human health. To determine how the two processes are coordinated throughout S phase, we characterize both processes together at high resolution. We find that transcription occurs during DNA replication, with transcription start sites (TSSs) not fully replicated along with surrounding regions and remaining under-replicated until late in the cell cycle. TSSs undergo completion of DNA replication specifically when cells enter mitosis, when RNA polymerase II is removed. Intriguingly, G2/M DNA synthesis occurs at high frequency in unperturbed cell culture, but it is not associated with increased DNA damage and is fundamentally separated from mitotic DNA synthesis. TSSs duplicated in G2/M are characterized by a series of specific features, including high levels of antisense transcription, making them difficult to duplicate during S phase.